Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

Abstract

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficileassociated disease is recognized. In this study, we identified Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects people and multiple animal species. Colonization of the gastrointestinal tract may be asymptomatic or cause a variety of disorders, including mild diarrhea, pseudomembranous colitis, and antibiotic-associated diarrhea (1). Recent outbreaks in North America and Europe document the seriousness of C. difficile infection (2). C. difficile toxins, including toxin A, toxin B, and a recently identified toxin, binary ADP-ribosyltransferase toxin C. difficile transferase, are thought to be the primary virulence factors that mediate C. difficile-associated disease (3). Because C. difficile colonizes gastrointestinal tissues and enterocyte-like Caco-2 cells (4, 5), colonization factors are also recognized as important virulence factors of C. difficile. A variety of colonization factors has been identified, including the following: capsule (6); proteolytic enzymes (7, 8); S-layer proteins P36 and P47 (9); adhesins such as SLPA, CCAP6, Fbp68, and 12-kDa protein (4, 9 -14); flagellins such as FliC and FliD (15, 16); and GroEL chaperones (17,18).Adhesion is the first step in bacterial infection, and several adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) 2 contribute to this step (19). MSCRAMMs located on bacterial surfaces mediate adhesion by binding to various extracellular matrices including fibronectin (Fn), laminin, collagen, elastin, and proteoglycan on host surfaces (19). Loss of some of these genes may attenuate virulence, indicating that MSCRAMMs are pivotal mediators of bacterial disease (20). Although a number of MSCRAMMs have been investigated in other bacterial pathogens, only a few clostridial adhesins, such as Fbp68, have been characterized, and the colonization mechanisms of C. difficile are still poorly understood (11).Manganese-binding proteins are important players in bacterial physiology by participating in cation homeostasis (21), carbon metabolism to promote nutrient acquisition (22), signal transduction (23), resistance to oxidative stress (24), and nutrient-deprived stress (25). In addition, the interaction of some bacterial adhesins and ECM components can be modulated by metal ions such as calcium (26,27). To date, the ability of manganese to mediate bacterial adhesion has not been reported.Previously, Fbp68 on the surface of C. difficile was shown to serve as an adhesin by binding to Fn, fibrinogen, and vitronectin (11). Interestingly, antibody to Fbp68 can be detected in sera from patients with C. difficile-associated disease, indicating that Fbp68 can induce a host immune response during C. difficile infection (28). Structural analysis of Fbp68 indicates t...

Novel Mycobacteria Antigen 85 Complex Binding Motif on Fibronectin

“…Fn is normally expressed on the surface of Caco-2 cells (32). Fn expression levels can be reduced by 62.7% with transfected Fn siRNA duplex as compared with cells transfected with negative siRNA duplex (Fig.…”

“…The k on , k off , and K D values are summarized in Table 4. Unlike the interaction of some bacterial adhesins and ECM components (32,34,37) Fig. 2A) were examined by using ELISA.…”

“…RNA duplexes were introduced into Caco-2 cells by the method of lipofection (31,32), and 8 ϫ 10 5 cells were transfected with 0.4 g of negative siRNA or Fn-siRNA. Adhesion assays were performed 72 h after lipofection.…”

Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization

“…It was speculated that L. casei probably utilizes other FbpA-independent mechanisms for attachment to the HT29 and Caco-2 cell lines or that the contribution of FbpA to binding in these models is small. In another recent report, a CDDFbp68 mutant was constructed in C. difficile 630 (insertion site at 102 bp) by using the same ClosTron gene knockout system (Lin et al, 2011). The authors assayed adherence to Caco-2 cells and showed that their mutant had similar fibronectin-and cell-binding activities to the wild-type strain.…”

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex

“…RNA duplexes were introduced into Caco-2 cells by the method of lipofection (38,39), and 5 ϫ 10 6 cells were transfected with 0.8 g of negative siRNA or elastin-siRNA. Adhesion assays were performed 72 h after lipofection.…”